Labeled antibodies are commonly used in immunoassays, such as ELISAs, in immunohistochemistry, and for identification of cellular components after gel electrophoresis such as the use of enzyme labeled antibodies in Western blot assays. The most common procedure is to detect the antigen with peroxidase-labeled specific antibody or first with unlabeled specific antibody followed with peroxidase-labeled anti-antibody. Addition of substrate, e.g., 3,3'-diaminobenzidine, results in a colored signal. In immunohistochemical experiments and in Western blots, the colored product is deposited at the site of the antigen. In cases in which the antigen is present at low levels, uncertain false negative results can be obtained. Accordingly, methods of amplifying the signal, without significant background "noise", have been sought.
Prior art immunoassays use peroxidase, frequently in the form of horseradish peroxidase, as a labeling enzyme coupled with a chromogen-hydrogen peroxide mixture as the substrate for detecting the antibodies. Further variations of the usual ELISA or EIA are disclosed in Sternberger, L. A., (1986) Immunocytochemistry: Third Edition, (Wiley and Sons); and Polak, J. M. and Van Noorden, S., (1983) Immunocytochemistry: Practical Applications in Pathology and Biology, Wright, pp. 214-215, wherein antibodies to peroxidase are used to detect peroxidase labeled antibodies bound to the molecule of interest. This method increases the specificity and sensitivity of the ELISAs.
Sternberger (1986) discloses that 3,3'-diaminobenzidine had been the preferred chromogen due to its high sensitivity and high insolubility, but was found to be carcinogenic and mutated samples. Further, McKimm-Breschkin, J. L., (1990) J. Immunol. Methods, Vol. 135, pages 277-280, discloses that although the preferred alternative has been 3,3',5,5'-tetramethylbenzidine (TMB), a non-mutagenic compound with similar properties, this compound when mixed with hydrogen peroxide in the reagent bottle breaks down, emitting color prematurely. Others, have disclosed that this premature reaction can be overcome by stabilizing the mixture with bacitracin, penicillin and N-methyl pyrrolidone. See U.S. Pat. No. 5,206,150, Tai, et al.; U.S. Pat. No. 4,891,314, Pauly, et al.; and U.S. Pat. No. 4,596,770, Parham, et al., the contents of which are hereby incorporated by reference.
The most widely used amplification procedure is through the use of peroxidase-labeled anti-peroxidase antibody, referred to as the "PAP" amplification. See, Sternberger, L. A., et al. (1970) J. Histochem. Cytochem., Vol. 18, pages 315-333. Sternberger (1970) disclosed that the maximum amplification possible by the PAP procedure was approximately 12-fold. Further amplification is prevented by steric interference.
However, unlike PAP amplification, the subject invention provides the potential for exponential amplification. This is accomplished by the use of an antibody specific for the product of the label-substrate reaction. Because enzymes continuously "turn over" substrate molecules into product, reaction with the molecules of product produced by a single enzyme molecule can result in marked amplification of the signal, well beyond that seen by PAP amplification or other known techniques.
Because of the enhanced sensitivity of this method, the invention provides for low level detection and the reduction of the occurrence of false negatives in all types of immunohistochemical detection. The invention further allows for the use of smaller amounts of antibodies directed to a target antigen, which antibodies may be in short supply or may be prohibitively expensive. The invention further provides an alternative to PCR detection of nucleic acids. These and other uses are more fully described below. Further uses apparent to those of ordinary skill in the art based on this description of the invention are deemed within the scope of the invention.